In vitro culture of lymphocyte colonies in agar capillary tubes after PHA-stimulation

Human peripheral lymphocytes were stimulated with phytohemagglutinin (PHA) in liquid culture, suspended in agar and incubated in glass capillary tubes. Compact colonies of lymphocytes were found growing along the tube bottom in a buffer film, while compact clusters and rare diffuse colonies were observed inside the agar. Several parameters affecting the clonal growth were studied and optimized: PHA-dose, agar concentration, gel length (volume), quantity and density of seeded cells per capillary and gel length. Colony yield mainly depends on the seeded-cell density with a sharp optimum at 2 X 10(5) cells/ml irrespective of gel length; higher cell densities reduce the colony yield, suggesting that colony growth is the result of both stimulatory and inhibitory factors produced by cooperating cells. Following the daily clonal growth was only possible with undisturbed tubes; the number of colonies steadily increased from day 2 until day 7. Densitometric colony scanning is possible, yet problematic. Colony yield (plating efficiency is 10--50-fold higher in agar capillaries than in the usual Petri dishes. An additional advantage is that the capillaries provide a basis for a simple and reliable assay system for determining regulatory factors of lymphocyte proliferation (including chalones).